This is a competitive renewal application to further characterize the molecular and cellular facets of host immunity in high-risk corneal transplants, distinguished by their rapid and high-frequency rejection. The goal of these studies is to gain new insights into the mechanisms that distinguish high-risk vs. low-risk transplant immunity. Our overarching hypothesis is that high-risk corneal grafts are characterized by a microenvironment which at once abrogates the tolerogenic potential of corneal antigen-presenting cells while also rendering the host regulatory T cells dysfunctional. To test this concept, we have generated specific hypotheses: 1. The subverted immune homeostasis in high-risk grafts can be restored by (a) expansion of host regulatory T cells (Tregs) through enhanced CD25/IL-2R signaling (Aim 1), and (b) generation of tolerogenic antigen-presenting cells in the graft (Aim 2); and 2. The defective Treg function in high-risk grafts overrides the physiologic angiogenic privilege of the cornea, thus further amplifying the immune response against the graft (Aim 3). To test these specific hypotheses, we will pursue the following specific aims: In Aim 1A, we will investigate the differential frequencies and functional characteristics of 'natural' and 'induced' Tregs in low- and high-risk grafts, and determine the molecular bases for Treg dysfunction that we have established in high-risk transplants. In Aim 1B we will determine the efficacy of amplified CD25 signaling through low-dose interleukin-2 treatment in expanding and restoring Treg function in high-risk grafted hosts. In Aim 2 we plan to continue our work enriching corneal donor buttons with tolerogenic antigen-presenting cells (tolAPC) through ex vivo conditioning, and to determine the effect of transplanting these tolAPC-enriched grafts on host sensitization, Treg frequency and function, and high-risk graft survival. In Aim 3 we will evaluate the contribution of adaptive T cell-mediated immunity to corneal angiogenesis and lymphangiogenesis by comparing the function of effector T helper-1 cells vs. Tregs in abrogating vs. promoting corneal angiogenic privilege. Our study design relies on using the expertise of our laboratory along with use of well-characterized mouse models of corneal transplantation in conjunction with in vitro immunological and cell proliferation assays, and use of transgenic mice permitting us to monitor the differentiation and fate of Tregs so as to gain mechanistic insights into the molecular regulation of corneal alloimmunity. The overall health relevance of this research is that corneal grafting represents the number one form of transplantation performed in the United States. However, while most high-risk corneal transplant patients rapidly reject their grafts, there has been no significant change in the management or prognosis of high-risk transplantation in decades. The long-term objective is to use the data derived from these aims to develop new strategies to promote high-risk graft acceptance without the use of systemic immunosuppressive regimens which can be highly toxic.